Immunoprecipitation of Reporter Nascent Chains from Active Ribosomes to Study Translation Efficiency

The study of translation is important to the understanding of gene expression. While genome-wide measurements of translation efficiency (TE) rely upon ribosome profiling, classical approaches to address translation of individual genes of interest rely on biochemical methods, such as polysome fractionation and immunoprecipitation (IP) of ribosomal components, or on reporter constructs, such as luciferase reporters. Methods to investigate translation have been developed that, however, require considerable research effort, including addition of numerous features to mRNA regions, genomic integration of reporters, and complex data analysis. Here, we describe a simple biochemical reporter assay to study TE of mRNAs expressed from a transiently transfected plasmid, which we term Nascent Chain Immunoprecipitation (NC IP). The assay is based on a plasmid expressing an N-terminally Flag-tagged protein and relies on the IP of Flag-tagged nascent chains from elongating ribosomes, followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) quantification of eluted mRNA. We report that elution of mRNA following IP can be achieved by treatment with puromycin, which releases ribosome-mRNA complexes, or with purified Flag peptide, which instead releases nascent chain-ribosome-mRNA complexes. In the example described in this protocol, untranslated regions (UTRs) of a gene of interest were used to flank a FlagVenus coding sequence, with the method allowing to infer UTR-dependent regulation of TE. Importantly, our method enables discrimination of translating from non-translating mRNAs. Additionally, it requires simple procedures and standard laboratory equipment. Our method can be used to test the effect of regulators, such as microRNAs or therapeutic drugs or of various genetic backgrounds, on translation of any user-selected mRNA. Key features • The novel NC IP protocol builds upon a previously published method for detection of mRNA-binding proteins (Williams et al., 2022). • The NC IP protocol is adapted for detecting mRNA actively undergoing translation. • The method uses mammalian cell culture but could be adapted to multiple organisms, including budding yeast (S. cerevisiae).

This protocol is used in: eLife (2023), DOI: 10.7554/eLife.87253.1 The study of translation is important to the understanding of gene expression.While genome-wide measurements of translation efficiency (TE) rely upon ribosome profiling, classical approaches to address translation of individual genes of interest rely on biochemical methods, such as polysome fractionation and immunoprecipitation (IP) of ribosomal components, or on reporter constructs, such as luciferase reporters.Methods to investigate translation have been developed that, however, require considerable research effort, including addition of numerous features to mRNA regions, genomic integration of reporters, and complex data analysis.Here, we describe a simple biochemical reporter assay to study TE of mRNAs expressed from a transiently transfected plasmid, which we term Nascent Chain Immunoprecipitation (NC IP).The assay is based on a plasmid expressing an N-terminally Flag-tagged protein and relies on the IP of Flag-tagged nascent chains from elongating ribosomes, followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) quantification of eluted mRNA.We report that elution of mRNA following IP can be achieved by treatment with puromycin, which releases ribosome-mRNA complexes, or with purified Flag peptide, which instead releases nascent chain-ribosome-mRNA complexes.In the example described in this protocol, untranslated regions (UTRs) of a gene of interest were used to flank a FlagVenus coding sequence, with the method allowing to infer UTR-dependent regulation of TE.Importantly, our method enables discrimination of translating from non-translating mRNAs.Additionally, it requires simple procedures and standard laboratory equipment.Our method can be used to test the effect of regulators, such as microRNAs or therapeutic drugs or of various genetic backgrounds, on translation of any user-selected mRNA.

Background
Translation is the process of protein synthesis using messenger RNAs (mRNAs) as template molecules, occurring in ordered steps (Blanchet and Ranjan, 2022), typically modulated by 5′ and 3′ untranslated regions (UTRs) (Hinnebusch et al., 2016;Mayr, 2019).The gold standard method for measurements of translation efficiency (TE) is ribosome profiling (Ingolia et al., 2009).However, this method allows for genome-wide analysis only, whereas, in some cases, specific investigation of TE of individual mRNAs might be of interest.A typical approach to understanding TE of specific mRNAs is to complement measurements of protein abundance with measurements of mRNA stability.Luciferase reporters are often used, with the luciferase coding sequence flanked by UTR(s) of interest.This approach, however, ignores influences of protein maturation and degradation rates.Alternatively, a measure of TE is provided by quantifying target mRNAs recovered after fractionation of polysomes or immunoprecipitation (IP) of ribosomal components.However, although highly translating mRNAs are loaded with a high number of ribosomes (Guttman et al., 2013;Ingolia et al., 2014;Liang et al., 2018), it is highly debatable whether a high number of ribosomes on transcripts is per se a robust indicator of high TE specifically of coding regions.Untranslated regions may instead be loaded with ribosomes actively engaged in translation (Ingolia et al., 2014); alternatively, transcripts may be bound by ribosomes while lacking coding capacity (Guttman et al., 2013).Additionally, neither approach provides information regarding which part of the endogenous mRNA is undergoing translation.This is relevant given that a multiplicity of mRNAs contain upstream open reading frames (Renz et al., 2020).Advanced imaging-and single cell-based methods have been developed (Biswas et al., 2019).Genetically encoded reporters enable visualization of translation but require co-transfection of multiple factors and intricate cloning for the addition of extensive features to reporter UTRs, e.g., arrays of stem-loops.Also, the requirement for sophisticated imaging platforms and complex data analysis are some other limitations of these recent methods.Here, we describe a simple yet effective alternative method that we recently introduced to assess TE of mRNAs of interest as dictated by UTRs (Cacioppo et al., 2023).Our assay is based on IP of Flag-tagged nascent chains, ensuring exclusion of non-translating mRNAs, which has already been proved an efficient approach (Raue et al., 2007;Aviner et al., 2013;Zhang et al., 2013).We have found the method particularly useful to measure the role of

Recipes
Critical: Prepare all solutions in RNase-free conditions.Note: All solutions should be prepared fresh.However, buffers can be stored at 4 °C, provided that some reagents are added just before the relative experimental step (denoted with "*").Transfer electroporated cells to 1.5 mL tube with 1 mL of warm medium.Gently invert tube and place at 37 °C.Caution: When pipetting electroporated cells into tube with warm medium, avoid contact between the pipette tip and the medium as this could cause arcing due to presence of FBS in the medium.12. Repeat step B11 two more times for the same condition.13.Before moving to the next condition, rinse the electroporation pipette tip in PBS multiple times.14.Centrifuge electroporated cells using MiniSpin at 300× g for 1 min.15.Resuspend in 1 mL of warm medium, plate in 10 cm dishes (one dish per condition) with final 10 mL of medium and incubate at 37 °C for 24 h.16.Clean and dispose of electroporation kit components according to the manufacturer's instructions.Critical: At sections C-F, strictly follow standard procedures for handling RNA.Treat work surfaces and pipettes with 70% ethanol and RNaseZap.Carry out all procedures on ice and always keep samples and solutions on ice.See General note 2.

C. Cell lysis
1. Prepare solutions.a. 35 mL of PBS + CHX 0.1 mg/mL b. 2.5 mL of lysis buffer c. 1.2 mL of TBS 1× 2. Add CHX at a final concentration of 0.1 mg/mL to electroporated cells and incubate at 37 °C for 15 min.3. Place plate on ice and wash cells once quickly with 10 mL of ice-cold PBS + CHX 0.1 mg/mL.4. Scrape quickly in 1 mL of ice-cold PBS + CHX 0.1 mg/mL while keeping plate on ice. 5. Transfer cells into 1.5 mL tubes and centrifuge using MiniSpin at 300× g for 5 min at 4 °C.6. Resuspend cell pellets in 200 μL of ice-cold lysis buffer and incubate on ice for 15 min.7. Centrifuge using MiniSpin at 12,000× g for 15 min at 4 °C.8. Transfer 180 μL of supernatants (Input) into new 1.5 mL tubes.

D. Immunoprecipitation
Note: Use 40 μL of beads slurry per condition.Beads need to be mixed to achieve a homogenous suspension.
Handle beads according to the manufacturer's instructions.Use a magnetic rack to separate beads from solutions.
1. Transfer 120 μL of beads slurry to a 1.5 mL tube.Remove solution in which beads were stored.

Data analysis
Translation efficiency (TE) is calculated using the percent input method, which compares the amount of target mRNA measured in the Elution fraction to the total amount of the target mRNA in the Input fraction.For this, first the ∆Cq is calculated as follows: where DF is dilution factor (e.g., if 5% of total Input is used for RT-qPCR, the DF is 20).TE of reporter mRNA is then calculated as: Representative data can be found in Figure 2.For each experiment, three technical replicates and three biological replicates should be performed.Appropriate controls for the experiment should be used, like using an untagged reporter protein (see Cacioppo et al., 2023) or untransfected cells.

Published: Sep 20
, 2023 a. Place medium, trypsin, and HBSS solutions at 37 °C.b.Place the electroporation tube into the holder in the Neon Electroporation machine.c.Pipette 3 mL of E2 electrolytic buffer from Neon transfection system kit into the electroporation tube.d.Prepare 3 × 1.5 mL tubes containing 1 mL of cell medium and keep at 37 °C.These are required later to enable recovery of cells following electroporation.e. Prepare 3 × 1.5 mL tubes each containing 3-5 μg of plasmid DNA.f.Prepare 3 × 1.5 mL tubes containing 1 mL of PBS.These are required later to rinse the electroporation pipette tip before moving to the next electroporation condition.g.Select the transfection program: voltage 1,150 V, width 30 ms, 2 pulses.h.Attach a gold electroporation tip to the electroporation pipette.2. Detach cells from 15 cm plates.a. Remove medium and wash cells in 10 mL of PBS.b.Remove PBS and add 1.5 μL of warm trypsin.c.Incubate at 37 °C for 3-5 min and then add 15 mL of warm medium.3. Transfer cells to a 50 mL Falcon tube.4. Centrifuge at 200× g for 3 min at room temperature.5. Wash cells in 10 mL of PBS and centrifuge again at 200× g for 3 min.6. Remove PBS and wash again in 1 mL of PBS, transferring cells to 1.5 mL tubes.7. Centrifuge using MiniSpin at 300× g for 1 min at room temperature.8. Remove PBS and equilibrate cells in HBSS by resuspending cell pellet in 1 mL of warm HBSS.9. Centrifuge using MiniSpin at 300× g for 1 min.10.Resuspend cells in 330 μL of warm HBSS and transfer into appropriate tube with plasmid DNA.Note: Electroporation is performed in three rounds per condition.For this, use 100 μL per electroporation round plus excess to avoid formation of bubbles during pipetting.Caution: Air bubbles could cause arcing during electroporation.11.Use the electroporation pipette to transfer cells to the Neon electroporation tube.Perform electroporation.

3 . 5 .
Add 750 μL of ice-cold lysis buffer and split equally into 3 × 1.5 mL tubes.4. Add 170 μL of Input samples to respective tube containing beads.5.Incubate tubes at 4 °C overnight gently rotating at 10-30 rpm.6. Prepare solutions.a. 3 mL of wash buffer.b. 600 μL of puromycin elution buffer or 600 μL of 3× Flag peptide elution buffer.7. Discard supernatants (flowthrough).8. Wash beads twice with 0.5 mL of ice-cold wash buffer.Note: Gently invert tube 4-5 times to wash beads.9. Perform elution.See General note 3.a.Elution by puromycin: add 200 μL of puromycin elution buffer to each tube containing beads and incubate at 4 °C for 30 min gently rotating at 10-30 rpm.Collect Elution samples and keep on ice for RNA isolation to be performed straight after.or b.Elution by 3× Flag peptide: add 100 μL of 3× Flag peptide elution buffer to each tube containing beads and incubate at 4 °C for 30 min gently rotating at 10-30 rpm.Collect eluates and repeat step.Collect eluates again and pool with previous respective eluates (total volume of elution samples 200 μL).Keep on ice for RNA isolation to be performed straight after.10.Perform RNA purification as in section E. Add a volume of H2O equivalent to the volume of RNA samples in a single well to the well corresponding to the no-template control reaction.Critical: Avoid formation of bubbles while pipetting during steps F3-F4.This might interfere with the amplification reaction.6. Cover plate with optical adhesive film.7. Quickly spin the plate to collect drops at the bottom of wells and to remove potential bubbles.8. Cover plate with darkening foil while setting up RT-qPCR machine.9. Load plate onto the RT-qPCR machine and start reaction.Perform one-step RT-qPCR according to the manufacturer's instructions.

Figure 2 .
Figure 2. Quantification by RT-qPCR of indicated reporter mRNAs in an experiment designed to test the role of different 3′ untranslated regions (UTR) configurations (D, L, S) of Aurora kinase A (AURKA), eluted via puromycin (left) or 3× Flag peptide (right).Results representative of three biological replicates.See Cacioppo et al., 2023 for details of the experiment.